Protein Kinase C Establishes the Signaling Boundary Between Mesodermal and Extraembryonic Lineages in Human Embryonic Stem Cell Differentiation

The interplay among mitogenic signaling pathways is crucial for proper embryogenesis. These pathways collaboratively act through intracellular master regulators to determine specific cell fates. Identifying the master regulators is critical to understanding embryogenesis and to developing new applications of pluripotent stem cells. In this report, we demonstrate protein kinase C (PKC) as an intrinsic master switch between embryonic and extraembryonic cell fates in the differentiation of human pluripotent stem cells (hPSCs). PKCs are essential to inducing the extraembryonic lineage downstream of various mitogenic modulators. PKC-alpha (PKCα) suppresses BMP4-induced mesoderm differentiation, and PKC-delta (PKCδ) is required for extraembryonic trophoblast cell fate. PKC activation overrides mesoderm induction conditions and leads to extraembryonic fate. In contrast, PKC inhibition leads to β-catenin activation, switching cell fate from extraembryonic to mesoderm lineages. This study establishes PKC as a central player directing the segregation of extraembryonic and embryonic lineages. The manipulation of intrinsic PKC activity could greatly enhance cell differentiation under mitogenic regulation in stem cell applications. Total RNA obtained from H1 hESC cultured in E6 medium supplemented with 20 ng/ml BMP4, 100ng/ml FGF2, 5 μM GF109203X, 3 mM 2DG, 100 nM LDN193189, 5 μM CHIR99021, 50 nM TPA, 1 μM PD0325901, 10 μM SB431542 and 5 μM DAPT. Samples were collected after 6 days.

促有丝分裂信号通路之间的相互作用，对于正常胚胎发生至关重要。这些通路通过细胞内主调控因子协同作用，以决定特定的细胞命运。鉴定这些主调控因子，对于理解胚胎发生过程以及开发多能干细胞（pluripotent stem cells）的新应用均具有关键意义。本研究证实，蛋白激酶C（PKC）是人类多能干细胞（hPSCs）分化过程中，胚胎细胞命运与胚外细胞命运之间的内在主开关。PKC家族成员在多种促有丝分裂调节因子下游诱导胚外谱系的过程中不可或缺。蛋白激酶C-α（PKCα）可抑制骨形态发生蛋白4（BMP4）诱导的中胚层分化，而蛋白激酶C-δ（PKCδ）则是胚外滋养层细胞命运形成所必需的。PKC的激活可突破中胚层诱导的培养条件，促使细胞向胚外谱系分化。与之相反，抑制PKC可激活β-连环蛋白（β-catenin），将细胞命运从胚外谱系转向中胚层谱系。本研究确立了PKC作为调控胚外谱系与胚胎谱系分离的核心因子的地位。在干细胞应用中，调控细胞内源性PKC活性可极大提升促有丝分裂调控下的细胞分化效率。本研究提取的总RNA来自于E6培养基中培养的H1人类胚胎干细胞（H1 hESC），培养基中添加了20 ng/ml 骨形态发生蛋白4（BMP4）、100 ng/ml 成纤维细胞生长因子2（FGF2）、5 μM GF109203X、3 mM 2-脱氧葡萄糖（2DG）、100 nM LDN193189、5 μM CHIR99021、50 nM TPA、1 μM PD0325901、10 μM SB431542以及5 μM DAPT。所有样本均在培养6天后收集。